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EPA Developing Risk-assessment Strategy for RNAi-based Pesticides

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As ag-bio giant Monsanto prepares to seek regulatory approval for a line of corn that uses RNAi to combat a key pest, the Environmental Protect Agency this week expressed concerns over the possible risks, both to humans and the environment, associated with plants that express gene-silencing dsRNA.

In order to develop a framework for assessing these risks, the EPA this week also held a meeting of agency scientists and outside researchers to solicit input on key concerns and questions surrounding the agricultural application of RNAi. A final report on the meeting's outcome is expected within 90 days.

As reported by Gene Silencing News, Monsanto is putting the finishing touches on a regulatory package for Smart Stax Pro — a corn strain designed to kill corn rootworms byexpressing widely used Bt proteins derived from the bacterium Bacillus thuringiensis along with dsRNA designed to silence an essential gene known as Snf7.

Ahead of Monsanto's regulatory submission, the EPA is now working to create guidelines for how it will assess the potential hazards of Smart Stax Pro and similar products, known as plant-incorporated protectants (PIPs).

According to a white paper issued by the EPA, one key area of concern for the agency are the possible effects of RNAi PIPs on humans and other mammals. Unlike non-PIP RNAi products, such as dsRNA-based pesticides that would be sprayed onto plants, the primary means by which PIPs would enter humans is through ingestion.

"Indications are that the majority, if not all, orally administered dsRNA is degraded by the acid environment of the stomach and the action of pancreatic RNases," which should "lessen the probability that any dsRNA PIP could survive gut passage and be taken up at a concentration that would trigger RNAi," the EPA said.

Questions remain as to whether chemically modified RNAi molecules could survive acid and enzymatic degradation in the gut, but there are a number of reports indicating that this is unlikely, including a paper co-authored by Monsanto researchers investigating a report of dietary microRNA uptake in humans, the EPA noted. In addition, the development of orally administered RNAi therapeutics has heretofore been unsuccessful, which further questions the dietary exposure and uptake of RNA molecules.

Perhaps the biggest area of worry for the EPA is the potential environmental impact of RNAi PIPs such as Smart Stax Pro. The agency noted that it has conducted ecological risk assessments for a number of PIPs over the past 20 years, most of which are related to plants expressing Bt proteins.

Although these past efforts can guide examinations of RNAi PIPs, "not all aspects of the fate of dsRNA in the environment and potential effects on non-target organisms are necessarily understood," it warned. "As a result, dsRNA PIPs may present unique challenges for ecological risk assessment that have not yet been encountered for other PIPs."

Key factors that influence the distribution of dsRNA into the environment and potential exposure to non-target organisms include the physical movement of PIP crop plant tissue, persistence of the RNAi molecule, and its movement once it is released from the plant, the EPA said.

Further, the agency said that it currently assumes that dsRNA expressed in a PIP plant "would be present wherever any of that crop plant tissue exists or is moved." For instance, on a crop field, such tissue would be present both above and below the soil surface where plants are grown, both in leaves and in roots.

However, it is not yet clear whether dsRNA from PIP plants would be present in root exudates, guttation droplets, or nectar, which would provide "additional on-field sources of non-target exposure," the EPA said.

As with Smart Stax Pro, RNAi PIPs typically target insects that chew and ingest plant tissue, and most exposure considerations have thus far focused on non-target insects that eat solid plant tissue. But recent studies examining the use of RNAi to control plant hoppers and other sucking insects suggest that gene-silencing molecules can be delivered via the phloem, making this and similar sources of exposure an area of risk assessment that should be considered going forward, the agency added.

Further, dsRNA from RNAi PIPs, once released from the plant, is expected to end up in the soil, where it could persist or be transported to other terrestrial or aquatic areas, the EPA stated. Once in aquatic environments, it may then move into sediment or exist in the water column.

Secondary exposure, wherein one organism consumes another that had previously ingested dsRNA, for instance, is another factor the EPA is weighing when it comes to RNAi PIPs, according to the white paper.

One recent study showed that dsRNA could be transferred from bees to Varroa mites, indicating that RNAi could be used as a way to protect the insects from the pests but also highlighting the possibility of secondary exposure.

While these kinds of animal and environmental exposure issues are important to consider with RNAi PIPs, the EPA noted that RNAi's unique mechanism of action raises challenges not present under existing risk-assessment paradigms.

Specifically, the ability of dsRNAs to silence genes with partial sequence homology to their targets or orthologs in other organisms creates the potential for "unexpected effects," the EPA said.

"Little information is available with which to estimate the likelihood of these effects resulting from unpredicted interactions of dsRNA with genes not intended for silencing," making them "significant factors affecting non-target risk," it added.

As such, those assessing the risks of RNAi PIPs should "remain cognizant of the possibility of unexpected effects that may not be readily predicted based on the known specificity for the target gene."

Additionally, RNAi PIP risk assessment should take into account the potential influence of latent effects on non-target organism testing, the EPA noted. Several toxicity studies submitted as part of the PIP regulatory process are short-term dosing studies, and the latency of effects with RNAi "may impact observations of both sublethal effects and mortality."

Also of importance is consideration of the appropriate life stage for testing. "Toxicity testing is often targeted toward young test organisms, as it is assumed that younger organisms are more sensitive to toxic substances than adults," the agency stated in the white paper. "However, sensitivity may not follow a specific trend over time, but may be the result of the importance of a target gene, or a gene affected by off-target silencing, in the organism at a specific life stage. … Given the range of possible unexpected effects, it is conceivable that an effect could occur in the field that would not be observed in the lab."

Lastly, RNAi presents the possibility of chronic effects, the EPA said. Current risk-assessment testing focuses on mortality as an endpoint, but off-target RNAi effects could results in deleterious effects that do not kill a non-target organism.

"Approaches other than non-target testing may provide additional information and reduce data requirements," the EPA stated. "Further development of predictive tools also has potential to reduce uncertainty as well as the scope of testing required to support a risk assessment."

Overall, the EPA expects that it could move ahead with risk assessment for RNAi PIPs based on current approaches, but doing so would require "conservative assumptions." Therefore, it will use the guidance provided during this week's meeting to help "reduce uncertainties in risk estimates."

Non-PIP RNAi

With Smart Stax Pro making its way toward the market, RNAi PIPs may be of immediate interest to regulators, but Monsanto and other companies are also working on RNAi-based pesticides that would be delivered topically. As a result, the EPA also touched on risk-assessment issues for such products in its white paper.

The agency noted that while it has some experience with RNAi PIPs, as with its approval of Smart Stax Pro field testing, it has not yet assessed the "hazards or risks of dsRNA applied directly to the environment as components of end-use products intended for pest control."

At the same time, the EPA has not received any application for the registration of a non-PIP dsRNA end-use product, it noted. But the agency anticipates that the risks of such products will be similar to those of RNAi PIPs, although the potential routes of direct and indirect exposure will be greater and include spray drift and surface runoff from foliage.

Additionally, pollenating insects such as honey bees could potentially carry dsRNA residues if they are present on plants at the time of application, and the accumulation of such residues could pose unanticipated effects among non-target organisms.

The EPA warned that current testing paradigms for non-PIP end-use products "likely will not be adequate" to assess the potential hazards of RNAi-based products resulting from off-target gene silencing, silencing of the target gene in unintended organisms, immune stimulation, and saturation of the RNAi machinery in cells.

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